Neurobrucellosis: laboratory features, clinical characteristics, antibiotic treatment, and clinical outcomes of 21 patients

Background Neurobrucellosis (NB) is a rare and serious complication of brucellosis. Its clinical manifestations vary, with no obvious specificity. At present, there is no clear clinical diagnosis or treatment for reference. In this study, we retrospectively analyzed the clinical data for 21 patients with NB to provide reference data for its further study. Methods We analyzed the epidemiological and clinical manifestations, laboratory tests, imaging examinations, cerebrospinal fluid, and treatment plans of 21 patients diagnosed with NB in the Department of Neurology, Xuanwu Hospital, Capital Medical University Beijing, China. Results The ages of the patients ranged from 15 to 60 years old (mean age 40.1 ± 13.33 years), the male: female ratio was 4.25:1. Thirteen patients had a history of animal (sheep, cattle) contact, three had no history of animal contact, and the contact status of four was unknown. Brucella can invade various systems of the body and show multi-system symptoms, the main general manifestations were fever (66.67%), fatigue (57.14%) and functional urination or defecation disturbance (42.86%). The main nervous system manifestations were limb weakness (52.38%) and hearing loss (47.62%).The main positive signs of the nervous system included positive pathological signs (71.43%), sensory abnormalities (52.38%), limb paralysis (42.86%). Nervous system lesions mainly included spinal cord damage (66.67%), cranial nerve involvement (61.90%), central demyelination (28.57%) and meningitis (28.57%). In patients with cranial nerve involvement, 69.23% of auditory nerve, 15.38% of optic nerve and 15.38% of oculomotor nerve were involved. The blood of eight patients was cultured for Brucella, and three (37.5%) cultures were positive and five (63.5%) negative. The cerebrospinal fluid (CSF) of eight patients was cultured for Brucella, and two (25.00%) cultures were positive and six (75.00%) negative. Nineteen of the patients underwent a serum agglutination test (SAT), 18 (94.74%) of whom were positive and one (5.26%) of whom were negative. A biochemical analysis of the CSF was performed in 21 patients, and the results were all abnormal. Nineteen patients underwent magnetic resonance imaging (MRI). Twenty-one patients were treated with doxycycline and/or rifampicin, combined with ceftriaxone, quinolone, aminoglycoside, or minocycline. After hospitalization, 15 patients improved (71.43%), two patients did not recover, and the status of four patients was unknown. Conclusions The clinical manifestations, CSF parameters, and neurological imaging data for patients with NB show no significant specificity or correlations. When patients with unexplained neurological symptoms accompanied by fever, fatigue, and other systemic manifestations in a brucellosis epidemic area or with a history of contact with cattle, sheep, animals, or raw food are encountered in clinical practice, the possibility of NB should be considered. Treatment is based on the principles of an early, combined, and long course of treatment, and the general prognosis is good.


Introduction
Brucella is a genus of Gram-negative bacteria that settle in cells and are commonly classified into sheep-type and bovine-type bacteria.The zoonosis caused by Brucella is called 'brucellosis' , which is also known as 'wavy fever' , 'Mediterranean fever' , or 'Malta fever' .It is an animal-specific infectious disease involving multiple systems, and is one of the commonest zoonoses in the world, with more than 500,000 new cases annually.It is prevalent in the Mediterranean Basin and countries in the Middle East, including Syria, Iraq, Iran, etc [1]...In China, it is mainly prevalent in pastoral areas, such as Inner Mongolia, Jilin, Heilongjiang, a Xinjiang, and Tibet, although cases occur in other provinces [2].It is transmitted to humans through contact with infected animals (cattle or sheep) or the consumption of uncooked meat products or unpasteurized dairy products.The clinical manifestations in these patients include fever, fatigue, weakness, hidrosis, joint pain, reduced appetite, headache, muscle pain, low back pain, hepatosplenomegaly, and arthritis [3].Neurobrucellosis (NB) is a rare complication of brucellosis, and its incidence in brucellosis patients varies between studies, but is usually < 10% [4][5][6].Brucella enters the blood through the reticuloendothelial system of the human body and causes bacteremia, then invading meninges with high affinity.When the host immunity declines, the bacteria proliferate, after which they invade other structures of the nervous system through the damaged bloodbrain barrier, resulting in various clinical symptoms, such as headache, limb weakness, hearing loss, meningitis, meningoencephalitis, myelitis, myelopathy, cranial neuritis, mental abnormalities, and vascular disturbances [7].Because the clinical symptoms of NB have no clear specificity, many of the laboratory tests usually used to diagnose brucellosis often produce negative results, so there is no clear clinical diagnostic standard or treatment method for NB at present.This leads to a low diagnosis rate and a high misdiagnosis rate in the clinical context, and it is difficult to distinguish NB from many other diseases of the nervous system, including tuberculous meningitis and multiple sclerosis [8].This necessarily poses great challenges for clinicians.
To improve the understanding of NB among clinical workers and to draw attention to it, in this study, we retrospectively analyzed the clinical data of 21 patients with NB, including its epidemiology, clinical manifestations, laboratory tests, imaging results, cerebrospinal fluid (CSF) parameters, and treatment plans, to provide reference data for further research into NB.

Methods
From January 2014 to August 2020, 21 patients diagnosed with NB were admitted to the Department of Neurology, Xuanwu Hospital, Capital Medical University (Beijing, China), including 17 males and 4 females, aged 15-60 years (mean age, 40.1 ± 13.33 years).All the 21 patients met all the following criteria These were [9][10][11] (1) clinical features consistent with known NB and an epidemiological correlation; (2) isolation of Brucella from blood or CSF; and/or positive serum agglutination test (SAT) (titres _> 1/160);and presence of anti-Brucella antibodies in CSF.(3) changes in the CSF, the increased WBC count, increased protein concentration, or reduced glucose concentration; (4) a more suitable alternative diagnosis could not be established.The basic information, epidemiological history, laboratory test results (including routine CSF parameters, CSF and serum Brucella cultures, CSF and serum Brucella serum agglutination tests, and imaging results), clinical symptom records, treatment plans, and prognoses of the 21 patients were collected and analyzed.The use of the above patient information in the study was approved by the Ethics Committee of the hospital, in one case, a 15-year-old patient also obtained consent from himself and his legal guardian.

Basic information
The ages of the 21 patients ranged from 15 to 60 years; one (4.76%)was < 18 years old and 20 (95.24%) were 18-60 years old (mean age, 40.1 ± 13.33 years).The age of NB onset was spread within the range of 18-60 years.Seventeen males and four females were enrolled, so the male: female ratio was 4.25:1.Therefore, the incidence rate was higher in males than in females.Eleven patients Twenty-one patients were treated with doxycycline and/or rifampicin, combined with ceftriaxone, quinolone, aminoglycoside, or minocycline.After hospitalization, 15 patients improved (71.43%), two patients did not recover, and the status of four patients was unknown.Conclusions The clinical manifestations, CSF parameters, and neurological imaging data for patients with NB show no significant specificity or correlations.When patients with unexplained neurological symptoms accompanied by fever, fatigue, and other systemic manifestations in a brucellosis epidemic area or with a history of contact with cattle, sheep, animals, or raw food are encountered in clinical practice, the possibility of NB should be considered.Treatment is based on the principles of an early, combined, and long course of treatment, and the general prognosis is good.
(52.38%) were from Hebei, two (9.52%) each from Inner Mongolia, Beijing, Henan, and Heilongjiang, and one (4.76%)from Shanxi, and the origin of one patient (4.76%) was unknown.Therefore, most patients came from northern China.Six patients (28.57%) had a history of contact with sheep (meat), 4 (19.05%) with cattle (meat) and sheep (meat), 3 (14.29%)with cattle (meat), and one patient (4.76%) had brucellosis in his family.Three patients (14.29%) had no history of animal contact and the histories of four (19.05%) were unknown.The incidence of NB was closely related to contact with cattle (meat) and sheep (meat) animals (Table 1).

Antibiotic treatments and clinical outcomes
Twenty-one patients were treated with doxycycline (0.1 g, twice a day) and/or rifampicin (0.45-0.6 g, once a day), combined with ceftriaxone (2 g, once a day), quinolones (e.g., 0.5 g of levofloxacin or 600 mg of ciprofloxacin, once a day), aminoglycosides (e.g., gentamicin 160,000 IU, once a day), or minocycline (100 mg, twice a day).According to the severity of the disease, 15 patients (71.43%) had improved or their CSF biochemical indices tended to normal or even completely normal after 2-27 days of hospitalization.However, two patients (9.52%) showed no obvious improvement and four patients (19.05%) had no follow-up treatment results (Table 4).

Discussion
Brucellosis is the most common infectious animal disease throughout the world, and in China, it is mainly seen in Inner Mongolia, Shanxi, Heilongjiang, Xinjiang Uygur Autonomous Region, Hebei, Liaoning, Jilin, and Ningxia Hui Autonomous Region.Most of the patients in this study were from Hebei, Inner Mongolia, Heilongjiang, and Shanxi, consistent with other epidemiological studies [2].However, four patients were from Beijing or Henan.With the development of tourism in pastoral areas and the migration of the labor force, the population mobility between pastoral areas and non-pastoral areas has increased greatly.Therefore, the incidence rate of brucellosis in non-pastoral areas shows an upward trend, as does the Brucella infection rate in the non-occupationally-exposed population [7].The most common sources of human brucellosis are sheep and cattle with brucellosis [1].Humans are infected by ingesting untreated milk or dairy products or by contacting infected animals, animal excreta, or meat, and the transmission routes include the lung, conjunctiva, skin, and digestive tract, since there are many pathogenic bacteria in raw milk, the risk of  infection by bacteria is very high, so this factor was also considered in the epidemiological investigation, but the medical records included in the study did not have the habit of taking raw milk, so there were no relevant medical records, and the rest is consistent with the etiology demonstrated in this study.It is also noteworthy that of one exceptional patient in this study, had a family member suffering brucellosis and the cause of family member was consumption of mutton with brucellosis infection, which had nothing to do with other reasons such as consumption of raw milk or dairy products or sexual intercourse.Therefore, in any epidemiological investigation of patients suspected of Brucella infection, we should also ask whether there is a recent history of travel to a pastoral area, a family history of brucellosis, or a history of contact with unusual pathogenic animals.The mechanism by which Brucella invades the central nervous system is still unclear.It is generally believed that Brucella causes bacteremia after entering the blood through the reticuloendothelial system of the human body, and then invades the meninges with particular affinity.When the host immunity declines, Brucella begins to proliferate and invades other nervous system structures [12].Recent studies have shown that Brucella induces tissue damage indirectly, most likely by activating the host's innate immune response after recognizing the corresponding receptor by Brucella antigen.Lipoprotein is an important brucella antigen that induces a variety of innate immune cells to secrete pro-inflammatory cytokines.Brucella nucleic acids have also been shown to be involved in the induction of inflammation.Astrogliosis and reactive microgliosis are unique signs of central nervous system inflammation associated with NB.Dysregulation of astrocytes and microglia caused by brucella infection creates a microenvironment in the central nervous system in which secretion of pro-inflammatory mediators leads to instability of glial structures, damage to the blood-brain barrier, and neuronal death.The immune mechanism of astrocyte hyperplasia and reactive microgliosis induced by Brucella is through promoting the secretion of inflammatory cytokines IL-6, IL-1β, TNF-α, and chemokines CCL2 and CXCL1 [13].The clinical manifestations of NB are varied, and the common clinical manifestations include fever, fatigue, myalgia, weight loss, splenomegalia, aphasia, vertigo, double vision, facial paralysis, hemiplegia, tremor, ataxia, and psychiatric symptoms [13,14].They also include meningitis, encephalitis, cerebrovascular diseases, subarachnoid haemorrhage, myelitis, radiculitis, cranial or peripheral nerve involvement, intracerebral and epidural abscess, demyelinating syndrome [11,13].The clinical manifestations observed in this study included fever, fatigue, limb weakness, hearing loss, defecation disorder, headache, unconsciousness, myalgia, weight loss, spastic paralysis of both lower limbs, hyperhidrosis, lethargy, breathing difficulties, and joint pain, among which systemic fever, fatigue and disturbed urination or defecation functions were common, together with nervous system symptoms such as limb weakness, hearing loss, unconsciousness and headache.Clinical examination often observed positive pathological signs, paresthesia, ataxia, eye movement disorder, and meningeal irritation signs, and the rate of meningeal irritation signs (1/21) was 4.76%, consistent with that reported in the literature (< 50%) [13].The most common clinical presentations of NB include meningitis, meningoencephalitis, myelitis, brain abscess, epidural abscess, radiculitis, cranial neuritis, demyelinating disease, and vascular disease, among which meningitis and meningoencephalitis account for 50% [12].Meningitis, the most typical clinical manifestation of NB, leads to irreversible central nervous system damage and is associated with neuronal damage.Overactivation of microglia leads to neurotoxicity and is one of the factors of neuronal injury in the central nervous system.Activated microglia can also secrete a variety of soluble mediators; however, most of the mediators produced are proinflammatory and neurotoxic [11].The cause of central nervous system inflammation and vasculopathy caused by NB may be the invasion of the central nervous system by Brucella, which interacts with astrocytes and microglia and induces the secretion of TNF-α, IL-1β and IL-6 by astrocytes and microglia.The induced inflammatory response will promote the activation of cerebral microvascular endothelium, the increase of vascular fluidity and the infiltration of immune cells [11].NB can involve one or more cranial nerves.Auditory nerve involvement is most common, and is generally considered to be caused by the involvement of the central auditory nerve pathway.The second most commonly involved nerve is the abducens nerve, which may be vulnerable to both direct and indirect injury because it has the longest intracranial run.The third most commonly involved nerve is the facial nerve, and NB can also damage the optic nerve [4].The most reliable criterion for the diagnosis of NB is a positive bacterial culture, but the positive rate of this method is low, and most patients also require a serum or CSF agglutination test to confirm their diagnosis [15].Blood cultures of Brucella were performed in eight patients, of which three were positive (37.50%) and five were negative (63.50%).CSF cultures of Brucella were performed in eight patients, of which two were positive (25.00%) and six were negative (75.00%).Of the 19 patients tested with SAT, 18 were positive (94.74%) and one was negative (5.26%).The positive rate of SAT was higher.The results of the CSF biochemical analysis were abnormal in all 21 patients tested.The CSF pressure was increased in 8 patients (40.00%), the WBC count was elevated in 21 patients (100.00%), the glucose in CSF to serum glucose ratio (%) was reduced in 21 patients (100.00%),IgG concentration and the protein concentration were elevated in 21 patients (100.00%), and the chlorine concentration was reduced in 14 patients (66.67%).Analysis of the CSF biochemical results showed that the CSF of NB patients was altered, but there was no obvious specificity, which was usually WBC, IgG concentration and protein concentration increased, glucose in CSF to serum glucose ratio (%) and chlorine concentration decreased, and may be accompanied by increased CSF pressure to different degrees.The changing trends in the WBC count, protein concentration, and glucose concentration are consistent with reports in the literature [16,17], but the incidence of reduced glucose concentration (66.67%) was higher than previously reported (1/3, 33.33%) [18].Leukocytosis in CSF may be due to Brucella activation of glial cells in the blood-brain barrier, and Brucella interaction with innate immunity in the central nervous system leads to increased transfer of phagocytic cells to the brain parenchyma [11].In 19 patients, MRI showed no obvious abnormalities in three patients but abnormalities in 16 patients.The main manifestations were white matter changes (42.11%), inflammatory changes (26.32%), vascular involvement (21.05%), and meningeal enhancement (15.79%).The first three manifestations have been reported in other research [3,19].In the present study, the lesions involved in the white matter were mainly concentrated around the lateral ventricle, temporal lobe, parietal lobe, cerebellar middle foot, and pons, which showed abnormal signals in the white matter.At present, the specific causes and mechanisms of abnormal white matter are unclear.It is thought that Brucella infection may activate the immune system, leading to demyelinating changes in the white matter [3].Meningeal lesions were concentrated in the temporal lobe, frontal part, and anterior skull base.
Because Brucella usually enters the cell cytoplasm for its survival and reproduction and most antibacterial drugs have weak penetration ability and must pass the blood-brain barrier, it is difficult for ordinary antibacterial drugs to achieve a satisfactory curative effect.Therefore, antibacterial drugs with good fat solubility and strong intracellular and central nervous system penetration should be selected for the treatment of NB.Because it is difficult to kill intracellular Brucella with a short course of antibacterial treatment, a long course of treatment and combined medications are recommended [7].Many studies have recommended antibiotics with good curative effects on NB, including doxycycline, rifampicin, streptomycin, compound sulfamethoxazole, and thirdgeneration cephalosporins, such as ceftriaxone or ciprofloxacin [11,20,21], and rifampicin, doxycycline, and ceftriaxone are considered standard drugs for NB [20].
In the present study, 21 patients were treated with doxycycline and/or rifampicin, combined with ceftriaxone, quinolones, aminoglycosamines, or minocycline.After hospitalization, 15 patients improved, two patients did not recover, and the status of four patients was unknown.The medication regimens were consistent with the medications recommended in the literature [11,20].The two patients who did not recover were treated in hospital for 2-3 days, which we consider a short course of treatment.Although the best course of treatment for NB is still uncertain, the results of this study are consistent with those of other reported studies [4], and suggest that patients treated early with combined medications have a good prognosis.
In other cases reported on NB [22][23][24], the clinical characters of NB were analyzed from 1 to 3 aspects including clinical manifestations, serum/cerebrospinal fluid agglutination test, imaging and cerebrospinal fluid laboratory examination.However, only case reports and retrospective clinical studies involving less than 13 patients were reported, and the results were similar to those of this study.However, due to the small number of cases included in the study, the characteristics of a single case are mainly introduced, and the comparison cannot be made from probability.A study to assess the prevalence of imaging abnormalities in patients with NB included 263 adult patients at 26 referral centers [25], but only the imaging abnormalities in patients with NB were analyzed, and the results showed that 118 (45%) patients with NB had abnormal neuroimaging findings.The main manifestations were inflammation (95.76%) > vascular involvement (66.95%) > hydrocephalus/cerebral edema (50.85%) > white matter involvement (33.05%).In this study, the 21 cases with abnormal imaging accounted for 78.95%, which was significantly higher than the above studies, including white matter changes (42.11%) > inflammatory changes (26.32%) > vascular involvement (21.05%) > meninges enhancement (15.79%).The reason for the different proportion of abnormal manifestations may be related to the number of included medical records, which need to be further confirmed by multi-center and big data research.Compared with the above existing clinical studies, the number of cases included in this study is larger, and the analysis and comparison from multiple aspects such as epidemiology, clinical manifestations, criteria for inclusion of cases, imaging and laboratory examination of cerebrospinal fluid, can provide a more comprehensive basis for the subsequent diagnosis of NB patients.
However, some limitations should be noted.First, the small number of cases.When the sample size is too small, the shortcoming is less of cases and only few of the culture positive to derive very valid information.Second, this article is a retrospective case analysis study, and there may be some uncontrollable factors in the implementation process, such as inconsistent detection methods, specimen inspection, or NB-related necessary examinations are not carried out, so it is necessary to carry out prospective clinical trials.

Conclusion
Brucellosis has always been a common and major health problem in developing countries, and NB is one of its serious complications.Although the incidence rate is usually < 10%, its early diagnosis and treatment are very important.In this study, we recorded and analyzed the clinical manifestations, laboratory and imaging results, and therapeutic outcomes of 21 patients with NB, to provide reference data for the diagnosis and treatment of this disease.Because NB is rare in the clinical context, there is no effective diagnostic "gold standard", and the clinical manifestations, CSF parameters, and imaging changes lack specificity, which make its diagnosis very difficult.Therefore, when patients present with nervous system dysfunction accompanied by fever and fatigue, or have a contact history with animals and animal products, the possibility of NB should be considered, and empirical treatment for NB should be given if necessary.It is challenging to develop a highly specific and highly sensitive diagnostic regimen for NB, and the development of such a test will be the focus of research into the diagnosis and treatment of NB.At present, there is no consistent treatment plan for NB.Although the reports of NB have increased in recent years, most results obtained by researchers are observational, and the number of cases is still small.Therefore, multicenter clinical trials of NB are required.

Table 1
Analysis of basic information of 21 patients with NB

Table 2
Analysis of clinical characteristics of 21 patients with NB

Table 4
Analysis of treatment plans and prognoses of 21 patients with NB